Fig. 2: MCFD2 interacts with FVIII through the canonical ligand-binding site of EF-hand proteins.
a 1H–15N HSQC spectra of [15N]MCFD2 with five molar equivalents of the ERGIC-53CRD-derived peptide (RRFEYKYSFKGPH) in the presence (red) and absence (black) of two molar equivalents of the FVIII-derived peptide (DPPSDLLLLKQSNSSKILVGRWHLASEK) (left). 1H–15N HSQC spectra of [15N]MCFD2 in the presence (green) and absence (black) of two molar equivalents of the FVIII-derived peptide (right). Proteins were dissolved in 20 mM MES (pH 6.0) containing 10 mM CaCl2, 150 mM NaCl, and 10% (v/v) D2O. NMR spectra were acquired at 303 K. b NMR chemical shift changes observed for MCFD2 in the presence (upper) and absence (lower) of the ERGIC-53CRD-derived peptide upon the addition of the FVIII-derived peptide. In the NMR perturbation profiles, residues for which the 1H–15N HSQC peaks could not be observed because of peak overlapping and/or broadening were denoted with asterisks, whereas proline residues were denoted with “P.” The observed chemical shift perturbations were quantified as Δδ = [(ΔδH)2 + (ΔδN/5)2]1/2, where ΔδH and ΔδN were the observed chemical shift changes for 1H and 15N, respectively. c The perturbed residues were mapped on a ribbon model of MCFD2 (upper). The dotted line indicates a disordered loop. A superimposed ligand-bound calmodulin complex (PDB code: 1CFF) is also shown (lower). The calmodulin and Ca2+-pump peptide ligand structures are colored in yellow and slate, respectively.
